Drying Biological Material

Mulyoto Pangestu, PhD1. Monash University Australia

2. Jenderal Soedirman University Indonesia

David Keilin, in his scholarly address in 1958 to The Royal Society reviewed several natural survival strategies including dehydration, cooling, lack of oxygen, and

increasing salt concentration,

and introduced term "Cryptobiosis" or now known as “Anhydrobiosis”.

Frontiers in Anhydrobiosis

Scientific observation on anhydrobisis has been started in early 18 century

• Leeuwenhoek (1702) discovered that certain ‘animalcules’ found in the sediments of roof gutters could be dried and then revived by the simple addition of water after being stored for his study in several months, – His finding as prompted considerable controversy,

but also much useful research.• Needham (1809) found similar effects with some

microscopic eelworms that infested wheat grains. These organisms, later identified as the larval stage of Anguillulina tritici, caused gall formation on the wheat, and in the dry state showed no sign of life or movement. Indeed, they were seen to crumble if touched while in this state, but were apparently restored to life if water was added

• Lazzaro Spallanzani (1803), Professor of Natural History at the University of Pavia, often credited with being the first to demonstrate survival of spermatozoa after cooling and rewarming, would not initially accept that organisms could undergo reversible desiccation. However, he eventually changed his mind and extended previous experiments by showing that dried rotifers could withstand heating up to 73C.

• Doy`ere (1842) demonstrated that, after thorough desiccation in a vacuum with sulfuric acid or calcium chloride, tardigrades and rotifers could withstand even higher temperatures, up to 125C.

Tardigrade

Artemia / sea-monkey

Artemia Merupakan kelompok udang-udangan dari phylum

Arthopoda, hidup di air asin dan toleran terhadap rentang

salinitas yang sangat luas, mulai dari nyaris tawar hingga

jenuh garam. Secara alamiah salinitas danau tempat mereka

hidup sangat bervariasi, tergantung pada jumlah hujan dan

penguapan yang terjadi. Apabila kadar garam kurang dari 6

% telur artemia akan tenggelam sehingga telur tidak bisa

menetas, hal ini biasanya terjadi apabila air tawar banyak

masuk ke dalam danau di musim penghujan. Sedangkan

apabilakadar garam lebih dari 25% telur akan tetap berada

dalam kondisi tersuspensi, sehingga dapat menetas dengan

normal.

Kista tertua artemia pernah ditemukan di sekitar Great Salt

Lake, diduga berusia sekitar lebih dari 10000 tahun

(berdasarkan metoda "carbon dating") dan masih bisa

menetas.

www.o-fish.com

www.o-fish.com

Siklus hidup artemia

Storage for Biological Materials

Frozen storageFrozen storage

• Advantages:– Has been proved by

many researchers– Produces live cells

and motile spermatozoa

• Disadvantages:– Requires access to liquid

nitrogen and appropriate storage tanks.

– Raises work safety issues– Accidental thawing is

detrimental– Possibility of cross

contamination– Limited storage capacity– COST

Frozen storage in liquid nitrogen is the standard protocol for storage of biological

material

Dry storageDry storage

• Significantly reduce the cost

• Allow unlimited storage capacity and save floor

space

• Circumvent all problems of cross contamination

• Allow genetic materials to be stored separately to

prevent use without consent.

• Significantly less likelihood of failure (eg.tanks)

• Eliminate all occupational health/building and

safety issues associated with liquid nitrogen

Dehydration is Destructive to living things.

But, when carried out in the Right Way it can

lead to long term preservation

• Why drying? Why drying? – Water is

highly reactive itself, may also contain remnants from the drug

manufacturing process including destructive enzymes such as proteases

– While in aqueous solution, these will be able

to degrade the product, but in the solid state produced by drying they are immobile

and harmless.

Freeze-dryingFreeze-drying

• Allows room temperature storage• Produces live and normal offspring

however, • Requires an expensive freeze-drying

machine and accessories or equipment

Freeze-drying is routinely used in food and pharmaceutical industries

Mouse sperm can be freeze-dried and stored at room temperature

(Wakayama and Yanagimachi, Nat. Biotech., 1998)

• Slow cooling– Extracellular ice formation from pure water in the

media– Increase osmolarity (as water turn into ice and left salt

behind)– Hypertonic effect– Slowly remove intracellular water– All pure water turn into ice– Cell completely dry (free of water)

Morphology, solubility and chemical integrity of the material can be maintained if water is

removed during frozen state.

From RT then slowly frozen

Pure water turn into ice and increase concentration of other molecules in remain suspension

Hypertonic effect, dehydration, CPA perme-abilises mem-branes

CPA replaces intracellular water

Cryoprotectant

Media (salt & water)

Ice

Intracellular components

• All extra cellular water (ice) sublimed/vacuumed out).

• Salts turn into crystal and

• Concentrated CPA but, may evaporate

• Dry specimen left behind.

Water removal

Vacuum to remove water

dry specimen

Vacuum pressure

Drying in the Right Way Important!!

to prevent damages and reduce destructive effects

Excipient (sugar)

Gen (?)

Water

Oxygen (?)

• Sugar molecules – produce stable solid products– form glassy matrix and preserve internal

structure– supporting tertiary structure

(polypeptide and protein)– preventing fro collapse when water

molecules are remove

• In the presence of sugar molecules, the process of drying becomes less damaging, and the solid product obtained is relatively stable.

• A small sugar called trehalose is found in many living things which have adapted to survive drying out.

Trehalose• has the ability to stabilize the cell during the drying

process making it possible to then rehydrate the cell and have it come back to full life.

• Trehalose is a naturally-occurring, glass-forming disaccharide (alpha-D-glucopyranosyl alpha-D-glucopyranoside), exploited by nature for millenia across a range of desiccation-resistant organisms.

• Found in the desert, the resurrection plant is a particular example, withstanding total drying for many years and coming back to life unharmed when re-hydrated.

• is an inert, non-reducing sugar, and does not interact with reactive molecules such as amino groups from peptides and proteins - preventing the degradation and aggregation which is common when drying with other excipients.

• is found in many of the foods we eat and is broken down to its glucose constituents by the human enzyme, trehalase.

Trehalose (Cont.)

Oxygen

• Oxidation/free radical damages– Membrane

– DNA

– Other cell components

– Impaired physiological system

Drying Mammalian (cells)

• No mammalian cells has been shown to synthesise or accumulate trehalose.– No cryotolerance– No anhydrobiosis– Can trehalose protect mammalian

cells against drying?

Incorporating trehalose into mammalian cells

• Genetic engineering using otsA and otsB genes (Guo et al, 2000)

• Reversible pores – genetically engineered mutant of Staphylococcus

aureus alpha-hemolysin to create pores in the cellular membrane. (Eroglu et al, 2000)

• Microinjection (Eroglu et al., 2002)

• Incubation (Guo et al., 2000; Wolkers et al., 2001)

• Electroporation (Shirakashi et al., 2002)

Methods &Reference

Cell type Treatments

Geneticengineering Guoet al., 2000

human embryonickidney cell line 293and human fibroblastcell line 12F

Cells were transfected with otsA and otsB from E. coliusing adenoviral vector. Air dry 72 h later by removingthe culture medium and storing cells at roomtemperature.Result: Viable and proliferate cells

IncubationPuhlev et al 2001

Human fibroblast Thermal shock 50mM T in DMEM followed byvacuum dry and storageResult: 60% viable day 1, 10% viable day 8.

IncubationMatsuo, 2001

Human cornealepithelial cells

0.2 M Trehalose in PBS followed by air drying for 30minResult: Viable cells after staining

IncubationWolkers et al.2001

Human platelets 52mM Trehalose in buffer at 37C followed by freezedrying.Result recovery >80% intact platelets.

IncubationGordon et al.,2001.

Human mesenchymalstem cells

50mM T; 3% Glycerol in hMSC medium 24h, vacuumstorage.Result: Up to 45% recovery of viable cells, butdecreased later.

Summary of reports on the use of trehalose in mammalian cell drying

Cell type Dryingmethod

Excipient Results References

Rat red bloodcells

Freeze drying PVP Recovered Meryman (1966)

Red bloodcells

Freeze drying Recover enzymaticfunction

Goodrich et al .(1992);Soewemimo-Coker

et al . (1993)

HES Recover enzymaticfunction

Rindler et al (1999a,b)

Fibroblast Air drying Trehalose Viable andproliferate cells

Guo et al (2000)

Cornealepithelial

Air drying Trehalose Viable cells Matsuo (2001)

Fibroblast Air drying Trehalose Viable cells Puhlev et al (2001)

Humanplatelets

Freeze drying Trehalose Recover enzymaticfunction

Wolkers et al .(2001b)

Humanmesenchymestem cells

Air dry andvacuum dry

Trehalose Recover viablecells but thenumber wasreduced by time

Gordon et al(2001)

Some attempts to preserve mammalian cells in the dry state.

Ucapan terima kasih

• Asian Development Bank

• Universitas Jenderal Soedirman

• Monash Institute Reproduction and Development

• National Institute of Health